Hepatitis C virus (HCV) often causes a prolonged and persistent infection in humans, and there is an urgent need for the development of a highly effective therapy and vaccine. Distinct genotypes and subtypes of HCV exist in different geographic locations. Infection with HCV genotype 1 (subtypes a and b) is prevalent in the USA, and is relatively resistant to available therapies. Recently, HCV growth in cell culture has been reported by us and other investigators. Majority of the work was done with HCV genotype 2a (JFH1 strain) in Huh7 cells or its derivatives. We have developed growth of HCV genotype 1a (H77 strain) in immortalized human hepatocytes (IHH). However, wild type HCV 1a yield is low and a significant sequence variation exists between HCV H77 and JFH1 strains. Although, HCV 1a particles are generated, virus growth from serial passages in hepatocytes has not been established. Therefore, it is extremely important to have a robust cell culture system for HCV 1a growth. Furthermore, titration of infectious virus particles is restricted with immunofluorescence or immunohistochemistry based assay. In this Developmental R21 grant application, we plan to generate a high titer HCV 1a by characterizing virus growth in IHH following different approaches, and develop a convenient readout for virus infection using a reporter assay. The results from the proposed work will help in further developing novel areas, including high throughput assay for antiviral screening with tremendous impact for drug and vaccine discovery programs. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) often causes a prolonged and persistent infection in humans, and there is an urgent need for the development of highly effective therapy and vaccine. We have recently generated HCV in cell culture. We will study to improve the in vitro growth of this virus and develop a sensitive readout for future use in high throughput antiviral screening and vaccine discovery programs.